Epithelial Ion and Fluid Transport Flashcards
1
Q
Why does water movement across epithelia matter
A
- body temperature regulation
- mucus movement (pathogen clearance from lung)
- renal fluid balance
- digestion and nutrient absorption
- reproduction
- diarrhoea (pathogen clearance from gut)
2
Q
Daily oral fluid input
A
2L
3
Q
Daily saliva fluid input
A
1.5L
4
Q
Daily gastric juice fluid input
A
2.5L
5
Q
Daily bile fluid input
A
0.5L
6
Q
Daily pancreatic fluid input
A
1.5L
7
Q
Daily intestinal fluid input
A
1L
8
Q
Daily total fluid input
A
9L
9
Q
How much fluid is lost in faeces
A
0.1L
10
Q
How much fluid is recovered by the small intestine
A
7L
11
Q
How much fluid is recovered by the large intestine
A
1.9L
12
Q
Action of cholera
A
- inhibits fluid reabsorption in gut
- epithelial function was measured by checking how much fluid was in bucket
13
Q
The transepithelial potential
A
- arises from ion movements
- ionic valency (z), concentration gradient (deltaCion), and ionic permeability (ease at which ion crosses membrane, Pion)
- ion movements are determined by fick’s law of diffusion
- to describe flux (J) over cell membrane, other terms are needed (Gion and Eion)
14
Q
Fick’s Law of Diffusion
A
Movement of flux (Jion) (moles.sec-1.cm-2) = Pion - deltaCion
15
Q
Problem with ficks law of diffusion
A
Pion is the product of the ion species and concentration gradient, and electrostatic attraction
16
Q
What is Gion
A
the ionic conductance of the ion across the membrane (amperes) -> measure of ionic movement
17
Q
What is Eion
A
the electrostatic diffusion potential (volts) -> measure of the size and direction of attracting forces
18
Q
Transepithelial potential of potassium
A
- as K+ ions leave cell across chemical gradient, deltaCK diminishes
- diffusion potential (EK) increases to retain K+ in the cell
- inward/outward movement of K+ depends on the membrane permeability of K+ (PK, determined by number of pumps/channels/transporters) and is measured by movement of charge (GK)
- when EK.EK = PK.deltaCK, net flux of K+ = 0
- value of transepithelial membrane potential (Em) at which equilibrium is established is given by Nernst equation
19
Q
Nernst Equation
A
- membrane potential at which equilibrium is established for a given ion
- Em = RT/zF ln(K[K+]o/[K+]i)
- Eion = 61log10([ion]o/[ion]i)
20
Q
Value at which there is no net flux of K+ ions at 37 degrees Celcius
A
Em = 61.log10([5]/[155]) Volts
Em = -0.092 Volts
Em = -92mV
21
Q
The Gibbs-Donnan Equilibrium
A
describes the effect of a non-permeable anion on transmembrane ion difference which drives water transport through osmosis
22
Q
How does water move across an epithelial membrane
A
2 routes:
- intracellular: movement of water occurs within cells and is regulated by water channels known as aquaporins
- paracellular: movement of water occurs between cells and is regulated by tight junction permeability
23
Q
What do we need for water movement across epithelial membrane
A
- osmotic gradient
- opening and closing of tight junctions
- aquaporins (channels specialised for the movement of water)
24
Q
Mechanism of fluid secretion
A
- presence of a sodium and potassium pump causes sodium to drive inward fluid uptake
- chloride is moved against gradient and accumulates inside the cell (membrane potential hyperpolarises)
- adding a chloride channel (CLCN) allows chloride to exit the epithelial monolayer and enter the apical membrane
25
Mechanism of fluid absorption
- opening of tight junctions createsan inward current
- sodium coupled glucose uptake helps to retain fluid in cases of diarrhoea
- epithelial sodium channel (ENaC) draws fluid across apical membrane and moves it towards the blood
26
Role of chloride in fluid secretion
- if ENaC dominates all epithelial membranes would dry and therefore we need secretion to go in the other direction using chloride
- chloride in blood plasma has negative charge so must be coupled with strong electrochemical movement of sodium and potassium to cotransport it across the basolateral membrane
27
Characteristics of Cl- channels epithelial cells
fluid secretion (basolateral -> apical transport)
28
Characteristics of Na+ channels epithelial cells
fluid absorption (apical -> basolateral transport)
29
Swelling-activated Cl- channel (IClvol)
- activated transiently by osmotic shock
- sustained opening does not occur
30
Calcium-activated Cl- channel (CaCC)
- activated by release of intracellular Ca2+ stores
- activity is transient
- unlikely to be sustained in development
31
Outwardly Rectifying Cl- Channel (ORCC)
- regulated by release of intracellular ATP
- maintains cell membrane potential by regulated depolarisation to physiological set point
32
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
- best characterised channel due to role in CF
- long presumed to be channel regulating fluid secretion in adult lung
33
Voltage-dependent Cl- channels (CLCN)
- recently characterised in lung
- expression pattern follows process of lung development
34
The CFTR
- member of ATP binding cassette glycoprotein superfamily
- 170kDa glycoprotein Cl- channel composed of 2 6-span transmembrane domains (pore), 2 nucleotide binding domains (NBD1 and 2), and a single R domain of highly charged AAs
35
The R domain of the CFTR
- a regulatory site containing several phosphorylation sites for protein kinases A and C
- activation of CFTR Cl- conductance requires ATP binding to the NBD domains and phosphorylation of the R domain
36
Role of NBD1 and NBD2 in CFTR
- hydrolyse ATP
- provides kinetic energy to open channel
37
Normal cycle regulation of CFTR
1) channel is inactive
2) cAMP activation of PKA phosphorylates R domain
3) ATP binds to NBD1&2
4) ATP is hydrolysed
5) channel opens and conducts Cl-
6) dephosphorylation of the R domain inactivates the channel
38
Loss of Phenylalanine 508 in NBD1 region of CTFR
- causes CF
- causes CFTR to be misfolded and sticks in ER, where it is broken down and never reaches membrane
- results in no fluid in airway secretion (dry lung), sticky mucus and pathogen colonisation
- treatment for CF reinstates the appropriate folding and translocates it back to membrane
- gene therapy treatments
39
Voltage-dependent Cl- ion channels (CLCN 1...7)
- widely distributed (found in epithelia, muscle, nerve tissue, also plants)
- channel opening is "gated" by membrane potential
- CLCN2 expressed in the epithelium is activated at negative (hyperpolarised) cell membrane potentials
- CLCN2 and CLCN3 are developmentally expressed in the foetal lung and control lung fluid volume during development
40
Structure of voltage-dependent Cl- ion channels (CLCN 1...7)
- 10 transmembrane domains which dimerise to form two pores
- each pore is voltage gated
41
Mutations in voltage-dependent Cl- ion channels
- CLCN1 mutation: myotronia (failure of muscles to relax after contraction -> cells remain depolarised)
- CLCN5 mutation: Dent's disease (fluid transport problems in the kidney resulting in kidney stones, calcium and protein loss in urine)
42
The ENaC channel
- found in all secretory epithelia
- composed of 3 subunits (alpha, beta, gamma)
- genetic knockout of alpha is lethal at birth due to flooding of lungs
- knockout of beta or gamma is not lethal but associated with reduction in rate of Na+ transport
- alphaENaC is the dominant pore-forming subunit
- association with beta and/or gamma is required to form a tetramer and confer Na+ selectivity
43
Structural domains of ENaC subunits
- cysteine-rich region on extracellular loop (rich in CSSC sulphur cross linking -> determines tertiary structure
- histidine glycine residue rich region involved in channel opening and closing
- proline tyrosine residue rich region which serves as a binding motif for NEDD4 (ubiquitin ligase which targets the subunit for membrane removal and proteolytic degradation)
44
Inhibition of ENaC
- Amiloride
- selective Na+ channel blockage shows high potency for ENaC blockers (benzamil and amiloride)
45
Activation of ENaC
- beta2 Adrenergic agonists (e.g. Isoproterenol)
- conductance is induced by catecholamines
46
Pseudohypoaldoseteronism (PHA)
- hereditary
- associated with resistance to aldosterone, leading to increased sodium excretion, dehydration, hypotension, hyperkalaemia, and metabolic acidosis
- disease is most evidence in kidney, sweat gland, pancreatic and salivary gland function
- can be lethal due to excessive hypotension and circulatory collpase
- results in excessive fluid retention in lung airways due to sustained Cl- secretion and inability to absorb Na+
- most lethal form caused by loss-of-function mutations in alpha, beta and gamma ENaC
47
PHA effect in renal system
- high Na/2Cl/K uptake
- high basolateral K+ secretion
- membrane removal and degradation
- Na+ secretion in urine
- ENaC channel recognized for degradation by NEDD4
48
Liddle's Syndrome
- hereditary
- characterised by salt-sensitive hypertension, hypokalaemia, metabolic alkalosis, and repressed aldosterone secretion
- results from gain-of-function mutations in C-terminal domain of beta or gamma ENaC which results in deletion of 45-75 amino acids from proline-tyrosine rich PY domain
- WT PY motif binds to NEDD4 (ubiquitin ligase) promoting internalisation and degradation of subunit
- loss of repressor activity = sustained Na+ absorption
49
Hypernatraemic effects of Liddle's Syndrome in Renal System
- ENac accumulation at apical membrane
- degradation of NEDD4 = sustained Na+ absorption
- increased Na+ pump activity compensates for Na+ uptake and causes Na+ secretion into blood with no route for removal
